Over the past few decades, the surge in the rates of obesity and type II diabetes in this nation has led to devastating health consequences. Stimulation of inducible mitochondrial thermogenesis, which wastes energy consumed as heat, is an attractive intervention strategy for obesity and related metabolic disorders. Uncoupling proteins (UCPs) are highly conserved thermogenic mediators that regulate inducible mitochondrial heat production in diverse tissues by controlling mitochondrial proton leak. In mammals, brown fat UCP1 is the established mediator of cold-induced thermogenesis. However, the low abundance of brown fat in a large percentage of adults challenges its thermoregulatory importance in these individuals and suggests that alternative thermogenic mechanisms exist. Skeletal muscle is also a key thermogenic organ in mammals, but whether UCPs are involved in muscle thermogenesis is unclear. We found that mice lacking the skeletal muscle-enriched UCP1 homolog UCP3 have sharply blunted (60- 100%) thermogenic responses to amphetamine-type stimulants, and the physiological fever inducers norepinephrine and lipopolysaccharide. In addition, mice specifically overexpressing UCP3 in muscle exhibit increased thermogenic responses. Fatty acids are essential activators of UCP1-3 induced proton leak, and therefore, thermogenesis. However, despite enormous efforts, the mechanistic basis for fatty acid activation of UCPs is unclear. Unsaturated, healthy fatty acids (e.g. oleate) are strong activators of UCPs relative to the more deleterious saturated analogs. Oleate and related fatty acids require auxiliary metabolic enzymes for full metabolism by beta oxidation. We found that UCP3 forms a novel, oleate- sensitive and functionally important interaction with 2,4, 3,5 dienoyl-CoA isomerase, a fatty acid metabolizing enzyme in the mitochondrial matrix whose substrates include many of the same fatty acids that bind and activate UCP3. Work in this grant will characterize the binding mechanisms and functional metabolic relevancy of this novel mitochondrial complex guided by three specific aims: Aim 1 - To define the structural and bioenergetic mechanisms regulating the binding of dienoyl-CoA isomerase and UCP3. Aim 2 - To establish the functional importance of dienoyl CoA isomerase for UCP3-induced mitochondrial uncoupling, and of UCP3 for the enzymatic activity of dienoyl CoA isomerase. Aim 3 - To explore the physiological functions of dienoyl CoA isomerase as a key regulator of lipid induced thermogenesis and fat catabolism in muscle and other UCP-relevant thermoregulatory tissues.